United States Prevention, Pesticides EPA712-C-98-097
Environmental Protection and Toxic Substances January 1998
Agency (7101)
&EPA Fate, Transport and
Transformation Test
Guidelines
OPPTS 835.5045
Modified SCAS Test for
Insoluble and Volatile
Chemicals
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INTRODUCTION
This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations.
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,
Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development
(OECD).
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U. S. Environ-
mental Protection Agency under the Toxic Substances Control Act (15
U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7U.S.C. I36,etseq.).
Final Guideline Release: This guideline is available from the U.S.
Government Printing Office, Washington, DC 20402 on The Federal Bul-
letin Board. By modem dial 202-512-1387, telnet and ftp:
fedbbs.access.gpo.gov (IP 162.140.64.19), or call 202-512-0132 for disks
or paper copies. This guideline is also available electronically in ASCII
and PDF (portable document format) from EPA's World Wide Web site
(http://www.epa.gov/epahome/research.htm) under the heading "Research-
ers and Scientists/Test Methods and Guidelines/OPPTS Harmonized Test
Guidelines."
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OPPTS 835.5045 Modified SCAS test for insoluble and volatile
chemicals.
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.} and the Toxic Substances
Control Act (TSCA) (15 U.S.C. 2601).
(2) Background. The source material used in developing this har-
monized OPPTS test guideline is 40 CFR 795.45 Inherent
Biodegradability: Modified SCAS Test for chemical Substances That Are
Water Soluble or Water Insoluble and Volatile..
(b) Introductory information—(1) Prerequisites, (i) Water solu-
bility of the test chemical must be established.
(ii) The organic carbon content of the test chemical must be estab-
lished.
(2) Guidance information, (i) Information on the relative proportions
of the major components of the test chemical will be useful in interpreting
the results obtained.
(ii) Information on the toxicity of the chemical may be useful to the
interpretation of low results and in the selection of appropriate test con-
centrations.
(3) Standard documents. This Test Guideline has been based on the
papers cited under paragraphs (e)(l) and (e)(2) of this guideline.
(c) Method—(1) Introduction, purpose, scope, relevance, applica-
tion and limits of test—(i) The method. (A) The method is an adaptation
of the Soap and Detergent Association Semi-Continuous Activated Sludge
(SCAS) procedure for assessing the primary biodegradation of
alkylbenzene sulphonate. The method involves exposure of the chemical
to relatively high concentrations of microorganisms over a long time pe-
riod (possibly several months). The viability of the microorganisms is
maintained over this period by daily addition of a settled sewage feed.
(B) Since the conditions provided by the test are highly favorable
to the selection and/or adaptation of microorganisms capable of degrading
the test chemical, the procedure may also be used to produce microbial
inocula adapted to selected chemicals for use in other tests. The test is
applicable to organic chemicals that are water insoluble or water insoluble
and volatile and that are not inhibitory to bacteria at the test concentration.
(ii) Reference chemicals. In some cases when investigating a new
chemical, reference chemicals may be useful; however, specific reference
chemicals cannot yet be recommended. Data on several chemicals used
in interlaboratory tests are provided (see following Table 1.) primarily so
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that calibration of the method may be performed from time to time and
to permit comparison of results when another method is employed.
Table 1.—Examples of Results of SCAS Test on Various Chemicals Used in the
OECD/EEC Intel-laboratory Test
Q Q Percent
Test chemical 0T (mg/L) fLn!\\ biodegradation/
(mg/Lj bioelimination
4-Acetylam inobenzene
sulfonate.
Tetrapropylenebenzene
sulfonate.
4-Nitrophenol
Diethylene glycol
Aniline
Cyclopentane tetracarboxylate ..
17.2
17.3
16.9
16.5
16.9
17.9
2.0
8.4
0.8
0.2
1.7
3.2
85
51.4
95.3
98.8
95.9
81.1
Duration of test is 40 days, except 120 days for cyclopentane tetracarboxylate.
(iii) Principle of the test method. (A) Activated sludge from a sew-
age treatment plant is placed in an aeration (SCAS) unit. The test chemical
and settled domestic sewage are added, and the mixture is aerated for
23 hours. The aeration is then stopped, the sludge is allowed to settle,
and the supernatant liquor is removed. The sludge remaining in the aer-
ation chamber is then mixed with a further aliquot of test chemical and
sewage and the cycle is repeated.
(B) This method requires use of a chemical-specific analytical tech-
nique or 14C-labeled test chemical. The purpose of the method is to deter-
mine the fate of the test chemical in a conventional activated sludge treat-
ment plant. To this end, a complete mass balance for the test chemical
is established by quantifying parent chemical in settled effluent sludge sol-
ids (insoluble test chemicals whether volatile or not), effluent plus solids
(insoluble test chemicals whether volatile or not), and off gases (volatile
test chemicals only). The identification and quantification of degradation
products in all phases are recommended, but not required.
(iv) Quality criteria—(A) Reproducibility. When primary biodeg-
radation is considered, very precise data are obtained for chemicals that
are extensively degraded. The results reported in the reference under para-
graph (e)(l) of this guideline suggest 95-percent confidence limits of less
than ±3 percent, and this includes interlaboratory tests. As would be ex-
pected, wider confidence limits are obtained for less biodegradable chemi-
cals.
(B) Possibility of standardization. Since the method uses a feed of
settled sewage, absolute standardization is not possible unless this feed
were replaced by synthetic sewage. However, since the method is designed
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to give an indication of the biodegradability potential of a chemical and
is not a simulation test such standardization is unnecessary.
(C) Possibility of automation. Automation of this method would be
possible but would be expensive. As the method is not labor intensive,
the exercise would offer few advantages.
(2) Description of the test procedure—(i) Preparations. (A) The
aeration units are cleaned and fixed in a suitable support. The air inlet
tubes are connected to the supply manifold. A small laboratory-scale air
compressor is used to aerate the units, and the air is presaturated with
water to reduce evaporation losses from the units.
(B) If the test chemical is volatile, exhaust gases from the aeration
units should be passed through a suitable trap (such as Amberlite
XAD-4, Rohm and Haas, Philadelphia, PA) to remove volatilized
organics.
(C) A sample of mixed liquor from an activated sludge plant treating
predominantly domestic sewage is obtained. Approximately 150 mL of
the mixed liquor are required for each aeration unit.
(D) The organic carbon analyzer is calibrated using potassium hydro-
gen phthalate.
(E) Stock solutions of the test chemicals are prepared: The concentra-
tion normally required is 400 mg/L as organic carbon which gives a test
chemical concentration of 20 mg/L carbon at the start of each aeration
cycle if no bio degradation is occurring.
(F) If the test chemical is insoluble in water at 400 mg/L it may
be necessary to use ultrasound dispersion to obtain a uniform stable sus-
pension. Alternatively, test chemical may be added directly to the aeration
units.
(G) The organic carbon content of the stock solutions is measured.
(ii) Test conditions. A high concentration of aerobic microorganisms
is used, and the effective detention period is 36 hours. The carbonaceous
material in the sewage feed is oxidized extensively within 8 hours of the
start of each aeration cycle. Thereafter, the sludge respires endogenously
for the remainder of the aeration period, during which time the only avail-
able substrate is the test chemical unless this is also readily metabolized.
These features, combined with daily reinoculation of the test when domes-
tic sewage is used as the medium, provide highly favorable conditions
for both adaptation and biodegradation.
(iii) Performance of the test. (A) A sample of mixed liquor from
a suitable activated sludge plant is obtained and aerated during transpor-
tation to the laboratory. Each aeration unit is filled with 150 mL of mixed
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liquor, and aeration is started. After 23 h, aeration is stopped, and the
sludge is allowed to settle for 45 min. The tap is opened, and 100 mL
of the supernatant liquor is withdrawn. A sample of settled domestic sew-
age is obtained immediately before use, and 100 mL is added to the sludge
remaining in each aeration unit. Aeration is started anew. At this stage
no test chemicals are added, and the units are fed daily with domestic
sewage only until a clear supernatant liquor is obtained on settling. This
usually takes up to 2 weeks, by which time the dissolved organic carbon
in the supernatant liquor at the end of each aeration cycle should be less
than 12 mg/L.
(B) At the end of this period the individual settled sludges are mixed,
and 50 mL of the resulting composite sludge is added to each unit.
(C) One hundred milliliters of settled sewage are added to the control
units, and 95 mL of settled sewage plus 5 mL of the appropriate test chem-
ical stock solution or suspension (400 mg organic carbon/L) to the test
units. If test chemical is added directly to aeration units, 100 mL of settled
sewage is added, as in the control units.
(D) Aeration is started again and continued for 23 h. The sludge is
then allowed to settle for 45 min and the supernatant drained off and ana-
lyzed for parent chemical. Before analysis the liquors are filtered through
washed 0.45 (im membrane filters and certifuged. Temperature of the sam-
ple must not exceed 40 °C while it is in the centrifuge.
(E) If the test chemical is insoluble or expected to sorb significantly
to sludge solids, settled sludge is also collected by an appropriate means
(such as centrifugation) and extracted to remove test chemical, and the
extract is analyzed for parent chemical.
(F) If the test chemical is volatile, traps for removing volatile organics
from exhaust gases are also extracted and the extracts analyzed for parent
chemical.
(G) The fill and draw procedure under paragraphs (c)(2)(iii)(C)
through (c)(2)(iii)(F) of this guideline is repeated daily throughout the test.
(H) Before settling, it may be necessary to clean the walls of the
units to prevent the accumulation of solids above the level of the liquid.
A separate scraper or brush is used for each unit to prevent cross contami-
nation.
(I) The length of the test for chemicals showing little or no biodeg-
radation is indeterminate, but experience suggests that this should be at
least 12 weeks.
(d) Data and reporting—(1) Treatment of the results, (i) The con-
centration of parent chemical in settled effluent sludge solids (insoluble
test chemicals whether volatile or not), effluent plus solids (insoluble test
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chemicals whether volatile or not), and off-gases (volatile test chemicals
only) is plotted versus time for the test units. As biodegradation is
achieved the level of the test chemical will decrease and approach a steady
state. Once the levels of the test chemical are found to be constant over
three consecutive measurements, three further measurements are made.
(ii) An example of the application of specific analytical technique
to the SCAS test is discussed in the reference in paragraph (e)(2) of this
guideline.
(e) References. The following references should be consulted for ad-
ditional background information on this test guideline.
(1) A Procedure and Standards for the Determination of the
Biodegradability of Alkyl Benzene Sulfonate and Linear Alkylate
Sulfonate. Journal of the American Oil Chemists Society 42:986 (1965).
(2) Games, L.M. et al. Fate and distribution of a quaternary ammo-
nium surfactant octadecyltrimethylammonium chloride (OTAC), in
wastewater treatment. Environmental Science and Technology 16:483-488
(1982).
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